Cloning
SARS-CoV-2 S HexaPro plasmid was a gift from Jason McLellan (Addgene plasmid # 154754)26. A mono-streptavidin sequence (mSA2, Kerafast, Accession ID: P22629) was inserted by restriction enzyme digest and ligation at the BstBI restriction site to make the spike construct.
The DNA sequence for AviTag-ferritin was ordered as a long single-stranded DNA oligo from Integrated DNA Technologies (IDT). The encoded protein, from N-terminal to C-terminal, consists of a His-tag, AviTag (GLNDIFEAQKIEWHE), a rigid linker (SLSTPPTPSTPPT), a bullfrog linker (Accession ID: P07797; residues 2–9 with N8Q mutation), followed by the Helicobacter pylori ferritin sequence (Accession ID: Q9ZLI1; residues 3-167 with I7E, N19Q, C31S mutations). Bullfrog linker and H. pylori ferritin sequence were adapted from a previous publication54.
Production, purification, and assembly of spike-based antigens
SARS-CoV-2 S HexaPro (spike-His) and mSA2-tagged-HexaPro (spike) plasmid was transfected, expressed, and purified as previously reported55. To prepare control spike protein which lacked mSA2, mSA2 from purified spike protein was removed by proteolytic cleavage at the internal HRV 3 C cleavage site.
Expression of AviTag-ferritin was carried out in E. coli strain CVB-T7 POL (Avidity, LLC), which overexpresses the E. coli biotin ligase BirA for in vivo biotinylation of Avi-tagged protein under the control of T7 promoter. The overnight culture of transformation colonies was diluted 200-fold into fresh Terrific Broth medium containing 0.1% MgSO4 and 0.04% glucose and incubated at 37° C with vigorous shaking. IPTG and biotin were added to the final concentration of 0.5 mM and 50 μm, respectively, when the OD600 reached 0.7. Expression and in vivo biotinylation were carried out at 20° C overnight. The harvested frozen pellet was resuspended in lysis buffer (50mM HEPES pH 7, 300mM NaCl, 30mM imidazole) with Roche complete protease inhibitor without EDTA. After lysis by sonication (Misonix S-4000), the centrifuge-cleared lysate was applied to Ni: NTA (Ni sepharose FF, Cytiva) pre-equilibrated with lysis buffer. Protein was eluted with 500 mM imidazole and desalted on a PD-10 desalting column.
To form the spike: ferritin nanoparticles, purified biotinylated AviTag-ferritin were mixed with at least a 100-fold excess of purified spike protein and incubated at room temperature for two hours. The sample was then fractionated on a Superose 6 increase 10/300 GL column, and the largest molecular weight peak was used for mouse inoculation experiments.
Liposomes used for liposome: spike were prepared in two steps. First, a 4:4:2 mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dioleoyl-sn-glycero-3-((N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl) nickel salt (DGS-NTA(Ni)) (Avanti Polar Lipids) were solubilized in chloroform and deposited as a thin film under nitrogen gas. Second, after rehydration in PBS, liposomes were extruded at a temperature above 60° C (the Tm of DSPC), with 30 passes through a 100 nm membrane with an Avanti Polar Lipids Extruder. The concentration of lipids was calculated according to established methods56.
To prepare liposome: spike immunogen, the liposome product was mixed with purified his-tagged spike protein in PBS with a final concentration of 4.7 nM and 1.1 µM, respectively. Ni: NTA beads were added to the sample, and the mixture was incubated for 1 h at room temperature to ensure no unbound spike-His remained in solution. Ni: NTA beads were removed by gravity filtration, and liposome: spike was further isolated by high-speed centrifugation at 4 ℃ and resuspended in PBS.
Spike rosettes were formed in a solution containing 20 µM NiCl2 and 0.55 µM of purified his-tagged spike protein incubated at room temperature overnight. The valency of spike rosette nanoparticles was estimated by visual inspection of negative-stain EM micrographs.
Characterization of liposome: spike
Size-exclusion chromatography of spike, liposome: spike, and liposome was carried out on a Superdex 200 column (GE Health Sciences) pre-equilibrated to PBS and run at 4 ℃ at 0.5 ml/min.
DLS measurements of liposome and liposome: spike were performed on the Zetasizer Nano. Samples of liposome, spike, or liposome: spike were filtered through a 0.22 μm Ultrafree-MC Centrifuge Filter (Durapore PVDF, Millipore) before loading to replicate preparation of vaccine samples. Although 0.22 μm is smaller than the peak diameter of liposome: spike as measured in this experiment, we reason that the liposome is flexible and can be deformed temporarily so as to fit through a narrow pore size. Buffer conditions (20 mM HEPES (pH 7.5) and 150 mM KCl) and cell holder temperature (25° C) were held constant during the measurement. Samples were placed at 25 °C for 15 min and then measured at the same temperature. All measurements were performed in triplicate, and an average value was calculated with standard error.
To perform Ni: NTA bead capture, sufficient beads were added to ensure full binding of the free spike-His in the sample. Liposome: spike was incubated with the beads for 1 h at room temperature before application to a gravity flow column and collection of flow-through. After washing with PBS, the bead-captured spike-His was collected with imidazole elution. The flow-through and elution were then analyzed by PAGE using a Novex 4–12% Bis-Tris acrylamide gel (Invitrogen). Bands were visualized with Coomassie staining.
Liposome: spike particle stability in 20% human serum in PBS was observed in the presence and absence of AddaVax adjuvant (InVivoGen). Temperature was kept at 37 °C for the duration of the experiment.
Electron microscopy of spike constructs
Samples for imaging by negative-stain electron microscopy were applied to glow-discharged carbon-coated copper EM grids and stained with uranyl acetate according to standard procedures. Grids were examined and imaged on a Talos L120C TEM operating at 120 kV acceleration voltage using a Ceta camera (Thermo Fisher Scientific).
Samples prepared for inspection by cryo-electron microscopy were spike or liposome: spike. 3 µL of each were applied to holey carbon grids (Quantifoil 1.2/1.3 Cu 300 mesh, Quantifoil Micro Tools) plasma-cleaned in a mixture of H2 and O2, blotted from 3 s, and plunge-frozen into liquid ethane using an FEI Vitrobot Mark IV at 7 °C with 98% humidity. Data were collected on the FEI Talos Arctica (200 kV) microscope equipped with a Falcon III camera (Thermo Fisher). Movies were recorded at a nominal 96,000x magnification (1.1 Å/px) over 40 frames in integrating mode with a total accumulated dose of 40 e−/Å2 and a defocus range of -1.0 to -2.5 μm. Image stacks were aligned and summed using motioncor257 with dose weighting, and CTF estimation was performed with Gctf58. Templates from the 2D classification of manually picked particles were used for autopicking in RELION 3.0 or cisTEM59. After template-based autopicking and removal of poor picks by 2D classification, an initial 3D model with C3 symmetry was generated. Particles were then aligned with 3D auto-refinement. Multiple rounds of 2D and 3D classification were used to remove poor particle picks further. The final 3D auto-refinement was performed using a soft mask, followed by postprocessing with the same mask.
Inoculation of mice and serum collection
Immunizations were carried out by intramuscular injection of female C57BL/6J mice (SLC) with 50 µL of inoculant containing a mix of 25 µL of antigen in PBS and 25 µL of AddaVax adjuvant. Each mouse was inoculated once on Day 0 and once on Day 15. Blood draws were carried out on Day 0 and Day 14 through the tail veil. After 1 or 2 months, the mice were euthanized by inhalation of carbon dioxide using a displacement rate from 30 to 70% of the chamber volume/min, and a final blood draw was carried out through the inferior vena cava. Serum was extracted from whole blood by centrifugation after room temperature incubation with a separating agent. The adjuvant used in all experiments was AddaVax (InVivoGen).
ELIspot
Peptide pools of SARS-CoV-2 S (JPT; PM-WCPV-S-1) were dissolved in DMSO (500 µg/mL). Freshly isolated splenocytes (2.5 × 105 cells per well) were stimulated with 1 mg/mL peptide solution or equimolar amounts of DMSO for 18 h. IFN-γ ELISpot assays were performed using Mouse IFN-γ Single-Color Enzymatic ELISpot kits (CTL; 2CT1B-mIFNGp-2M2), according to the manufacturer’s protocol. CTL ImmunoSpot S6 Analyzer was used for spot counts and determined by subtracting background spot counts in a DMSO-treated well from a peptide-treated well for antigen-specific spot counts.
Animals
Female 6–8-week-old, specific pathogen-free C57BL/6J mice were obtained from Japan SLC. Protocols of all mouse experiments were performed in accordance with the relevant guidelines and regulations as approved by the Animal Care and Use Committee at the Okinawa Institute of Science and Technology Graduate University (approval number 2020-295). Golden Syrian hamsters, specifically pathogen-free, female, 3 weeks of age, were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Food and water were given ad libitum. All hamster experiments were performed in accordance with the relevant guidelines and regulations as approved by the Tottori University Animal Care Committee (approval number 21-Y-46). This study was performed according to the ARRIVE guidelines.
Vaccine administration to hamsters
After one week of acclimation, the animals received the first vaccine dose via the intramuscular (i.m.) route. Animals were anesthetized by isoflurane inhalation before vaccination. Each hamster was held in one hand while the vaccine was injected into the deltoid muscle with a 23G needle. Either spike protein (N = 4) or liposome: spike (N = 4) were inoculated at 3 µg/ 50 µL volume (1:1 of antigen: adjuvant AddaVax volume) per animal. Control animals were inoculated with 50 µL saline (i.m.) (N = 4). Animals received the second vaccine dose via the same route two weeks after the first dose.
Serum isolation from hamsters
Pre-immune sera were isolated from the retro-orbital blood before the first vaccination. Post-immune sera were separated before the second vaccination and 1 week after the second vaccination. All sera were kept at -80 °C until analysis.
Virus infection
Two weeks after the second dose of vaccine, animals were anesthetized first with isoflurane inhalation, followed by injection of 1 mL fluid anesthesia (0.03 mg/mL medetomidine, 0.4 mg/mL midazolam and 0.5 mg/mL butorphanol) via subcutaneous (s.c.) route. Upon confirming the loss of the pedal withdrawal reflex, animals were inoculated with SARS-CoV-2 (0.2 mL, 105.5 TCID50/0.1 mL; strain SARS-CoV-2 WK-521, GISAID ID: EPI_ISL_40866760) via the intranasal (i.n.) route. SARS-CoV-2 was provided by the National Institute of Infectious Disease (NIID, Tokyo, Japan), propagated with VeroE6/TMPRSS2 at the BSL3 lab of Tottori University, and used at passage 9. Weight was measured and recorded daily until 14 days post-infection (DPI).
ELISA
50 µL/well of 2–4 µg/mL of spike was applied to Immulon 4 HBX plates and adsorbed at 4° C overnight. The next day, plates were washed with 200–300 µL of PBS + 0.1% Tween-20 (PBS-T) and blocked with 200 µL PBS-T + 3% milk solution for 1 h at 20 °C. After blocking, block solution was thrown off, 3x or 4x serial dilutions of serum samples in PBS-T + 1% milk were added at 100 µl /well, and plates were incubated for 2 h at 20 °C. After incubation, plates were washed with 200–300 µL of PBS-T and 100 µL of 1:3000 diluted goat anti-mouse IgG(Fab specific)-peroxidase antibody (Sigma) or goat anti-hamster IgG(H + L)-HRP antibody (Southern Biotech) in PBS-T + 1% milk was applied and incubated on the plates for an additional hour. Finally, plates were washed with 200–300 µL of PBS-T, and the bound antibody was detected with SigmaFast OPD solution according to manufacturer instructions. Each sample was tested in duplicate. Mean intensities were plotted against the log of reciprocal serum dilution factor in GraphPad Prism v8 and fitted with a sigmoidal, four-parameter logistic nonlinear regression. Endpoint titers are defined as the dilutions for which the calculated curves were equal to 0.2 absorbance units.
Production and titration of CoV-2 spike protein-pseudo-typed recombinant VSV
HEK-293T/17 (NIBSC CFAR catalog: #5016) cells grown in DMEM + GlutaMAX + 10% FCS + 1% v/v penicillin-streptomycin and at ~ 80% confluency in 10-cm culture dishes were transfected with 15 µg pCAGGS SARS-CoV-2 spike plasmid (NIBSC CFAR catalog: #100976) mixed with 60 µg of PEI transfection reagent using typical methods and incubated overnight at 37° C with 5% CO2 for protein expression. The morning of the next day, cell media was carefully removed, and cells were infected with G*ΔG-luc rVSV (KeraFast) at MOI of 0.1 in 5 mL of serum-free DMEM for 2 h at 37° C. After incubation the plate was washed 3 times with 3 ml PBS and left in 8 mL of DMEM + GlutaMAX + 10% FCS + 1% v/v penicillin-streptomycin for 24 h at 37 °C, 5% CO2. Cell culture supernatant was collected the next day, passed through a 0.45 μm filter, and frozen in 200 µL aliquots on dry ice.
Infection of VeroE6/TMPRSS2 cells (JCRB1819) by titrations of CoV-2 spike protein-pseudo-typed recombinant VSV was performed in 96-well plates. VeroE6/TMPRSS2 cells grown in a 10-cm culture plate to 90–100% confluency were detached with Trypsin-EDTA, and the cell suspension was diluted 6-8x to prepare sufficient volume for transfer of 100 µL/well into a 96-well culture plate. Cells were allowed to adhere to the plate for at least 2 h at 37° C, 5% CO2. After confirming cell adhesion, a 4-fold dilution series of CoV-2 spike protein-pseudo-typed recombinant VSV starting with 4x diluted virus stock in media was used to infect the cells at 100 µL/well in quadruplicate. Pseudovirus was incubated with cells for 24 h at 37° C, 5% CO2. The next day, infection was assessed using the Bright-Glo Luciferase Assay system (Promega) and measured on a DTX800 multimode plate reader (Beckman Coulter, Indianapolis, IN, USA). A viral dilution achieving, on average, 10,000–30,000 RLU was selected for use in pseudovirus neutralization assays.
Pseudovirus neutralization assays
Pseudovirus neutralization assays were carried out similarly to the virus titration experiment described above, but prior to infection, serial dilutions of sera samples in media were mixed with pseudovirus at 2× the selected viral dilution in 96-well plates. In parallel, one column of 2× diluted virus was diluted with an equal volume of media, and one column was filled with 120 µL/well of media only. The sera-pseudovirus plate was incubated for 1 h at 37° C, 5% CO2 before adding 100 µL of each well into the corresponding position of a 96-well plate containing adherent VeroE6/TMPRSS2 cells (prepared as described above). After 24 h at 37 °C, 5% CO2 infection was detected using the BrightGlo Luciferase Assay system, and %neutralization was calculated using the formula below, where RLU is the relative luciferase units of a given well, RLUcell, max is the maximum RLU of the cells mixed with media only, and RLUvirus, min is the minimum RLU of cell infection with pseudovirus in the absence of serum.
$$\:\%neutralization=100\%\times\:\left(1-\frac{RLU-{RLU}_{cell,max}}{{RLU}_{virus,min}}\right)$$
Each dilution series of serum was tested in duplicate or triplicate. The mean %neutralization for each dilution was plotted against the log of reciprocal serum dilution factor in GraphPad Prism v8 and fitted with a sigmoidal, four-parameter logistic nonlinear regression. The calculated equation was constrained to have a top value of 100% and a bottom value of 0%.
